1. Field of the Invention
The invention relates to methods of producing therapeutic proteins that interact with Fc receptors, e.g., antibodies, wherein the composition of the oligosaccharide chains are optimized for avidity of the antibody for its target as well as the Fc receptor binding affinity thereby optimizing the effector function activity of said antibodies as compared to nonoptimized methods of producing glycosylated antibodies.
2. Description of the Related Art
Antibodies are soluble serum glycoproteins that play a significant role in innate immunity. The carbohydrate structures of all naturally produced antibodies at conserved positions in the heavy chain constant regions varies with isotype. Each isotype possesses a distinct array of N-linked oligosaccharide structures, which variably affect protein assembly, secretion or functional activity (Wright, A., and Morrison, S. L., Trends Biotech. 15:26-32 (1997)). Referring to FIGS. 1 & 2, the structure of the attached N-linked oligosaccharides varies considerably, depending on the degree of processing, and can include high-mannose, as well as complex biantennary oligosaccharides with or without bisecting GlcNAc and core Fucose residues (Wright, A., and Morrison, S. L., supra). Typically, there is heterogeneous processing of the core oligosaccharide structures attached at a particular glycosylation site such that even monoclonal antibodies exist as multiple glycoforms. Likewise, it has been shown that major differences in antibody glycosylation occur between antibody-producing cell lines, and even minor differences are seen for a given cell line grown under different culture conditions.
Sialic acid on glycans (static groups) are known to be important in prolonging the serum half-life of glycoproteins other than antibodies (Stockert, R. J. (1995) Physiol. Rev. 75, 591-609). Thus far, the role of sialic acid on monoclonal antibodies (Mabs) is not well understood. The serum half-life of Mabs is particularly long-lived and construction of Fe-fusion proteins has proved a useful strategy in developing therapeutic proteins, e.g., the protein enteracept.
Antibodies and T-cell receptor molecules possess regions that are responsible for specific cell surface receptor binding, which binding modulates the cellular response. In the immune system, these functions are classified as humoral and cellular. Antibodies are often referred to as adaptor molecules linking humoral and cellular immune mechanisms: humoral responses being attributed mainly to mature, secreted, circulating antibodies capable of high affinity binding to a target antigen. Cellular responses are attributed to the consequences of cellular activation by binding of ab-ag complexes and by downstream sequelae caused by the release of cell mediators as a result of ab-ag complex binding to effector cells. These cellular responses include neutralization of target, opsonization and sensitization (if antigen is displayed on the surface of a cell), sensitization of mast cells, and activation of complement. For cellular targets, that is, cell surface antigens, these effector functions lead to what is commonly known as antibody-dependent cellular cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC).
Among antibody isotypes (e.g., IgE, IgD, IgA, IgM, and IgG), IgGs are the most abundant with the IgG1 subclasses exhibiting the most significant degree and array of effector functions. IgG1-type antibodies are the most commonly used antibodies in cancer immunotherapy where ADCC and CDC activity are often deemed important. Structurally, the IgG hinge region and CH2 domains play a major role in the antibody effector functions. The N-linked oligosaccharides present in the Fc region (formed by the dimerization of the hinge, CH2 and CH3 domains) affect the effector functions. The covalently bound oligosaccharides are complex biantennary type structures and are highly heterogeneous (see FIGS. 1 and 2). A conserved N-linked glycosylation site at Asn297 lies in each CH2 domain. In the mature antibody, the two complex bi-antennary oligosaccharides attached to Asn297 are buried between the CH2 domains, forming extensive contacts with the polypeptide backbone. It has been found that their presence is essential for the antibody to mediate effector functions, such as ADCC (Lifely, M. R., et al., Glycobiology 5:813-822 (1995); Jefferis, R., et al., Immunol Rev. 163:59-76 (1998); Wright, A. and Morrison, S. L., supra).
The heterogeneous oligosaccharides decorating the Fe-portion antibody or antibody-derived structures comprising produced by various host cells contain predominantly sialic acid, fucose, galactose and GlcNAc residues as terminal sugars (Raju, T. S., et al. Glycobiology 2000. 10(5): 477-86). It has been shown that some of these terminal sugars, particularly exposed galactose, core fucose and bisecting GlcNAc residues, affect the structure of the Fc-portion of the molecule and thereby alter antibody effector functions. Effector functions such as ADCC activity and CDC activity which relies on binding to cell surface receptors known as Fc-receptors, as well as the binding to various ligands including C1q complement protein can be altered by the composition of the appended glycan (Presta L. 2003. Curr Opin Struct Biol. 13(4):519-25). The majority of the N-linked glycans attached at the Fc are not sialylated to a significant extent (Idusogie E E, et al. 2000. J Immunol. 15:164(8):4178-84).
The major structures found in human IgG and other recombinantly-produced IgGs are the complex biantennary structures with or without exposed Gal residues (FIG. 1). There are a number of mammalian host cells that are currently used to express recombinant antibodies for research purposes, as well as, biopharmaceutical production. Host cell species of origin as well as culture conditions can cause the extent and structure of glycans appended to recombinantly expressed molecules to vary. Two commonly used host cell lines for the recombinant expression of antibodies are Chinese hamster ovary cells (CHO) and mouse myeloma cells (sp2/0, 653, NS0). While CHO cells express recombinant antibodies which are virtually devoid of sialic acid glycan the glycans are 99% fucosylated. The presence of fucose has been shown to be a major contributor to reduced Fe-gammaIII receptor and therefore ADCC. Mouse myeloma cells express recombinant antibodies with up to 50% sialic acid but with generally less fucose. As stated above, these differences can have significant effects on antibody activity in vivo.
Therefore, it would be desirable to be able to reduce sialylation of glycans associated to therapeutic antibodies in a manner which eliminates the need for post-harvest processing and at the same time provides a reasonably homogeneous structure with respect to sialic acid content.